A stem cell must meet the following criteria: (1) ability of a clonal stem cell population to self-renew; (2) ability of a clonal stem cell population to generate a new, terminally differentiated cell type in vitro; (3) ability of a clonal stem cell population to replace an absent terminally differentiated cell population when transplanted into an animal depleted of its own natural cells.
The neonatal period in human development is characterized by the presence of “stem” cells with the potential to develop along multiple differentiation pathways. The terminal differentiation of these cells is determined by cytokine and hormonal cues which co-ordinate organogenesis and tissue architecture. Murine embryonic stem (ES) cells have been isolated and studied extensively in vitro and in vivo. Using exogenous stimuli in vitro, investigators have induced ES cell differentiation along multiple lineage pathways. These pathways include neuronal, B lineage lymphoid, and adipocytes (Dani et al., 1997, J. Cell Sci. 110:1279; Remoncourt et al., 1998, Mech Dev 79:185; O'Shea, 1999, Anat. Rec. 257:32). The ES cells have been manipulated in vivo by homologous recombination techniques to generate gene specific null or “knock-out” mice (Johnson, 1989, Science 245:1234). Once ES cell clones lacking a specific gene are isolated, they are transplanted into a fertilized murine zygote. The progeny of this isolated ES cell can develop into any and all murine tissues in a coordinated manner.
Multipotential stem cells exist in tissues of the adult organism. The best characterized example of a “stem cell” is the hematopoietic progenitor isolated from the bone marrow and peripheral blood. Seminal studies by Trentin and colleagues (Trentin, 1965, Cardiovasc. Res. Cent. Bull 4:38; Till & McCulloch, 1961, Rad. Res. 14:213) examined lethally irradiated mice. In the absence of treatment, these animals died because they failed to replenish their circulating blood cells; however, transplantation of bone marrow cells from syngeneic donor animals rescued the host animal. The donor cells were responsible for repopulating all of the circulating blood cells. A wealth of elegant studies have gone on to demonstrate that donation of a finite number of undifferentiated hematopoietic stem cells is capable of regenerating each of the eight or more different blood cell lineages in a host animal. This work has provided the basis for bone marrow transplantation, a widely accepted therapeutic modality for cancer and inborn errors of metabolism. Thus, hematopoietic stem cells remain present in normal human bone marrow throughout life; they are not limited to the neonatal period.
There is exciting new evidence that hematopoietic progenitors may not be limited to the bone marrow microenvironment. Investigators at the University of Calgary have examined neuronal stem cells, which routinely differentiate along neuronal cell lineage pathways. When these cells were transplanted into lethally irradiated hosts, the investigators detected the presence of donor cell markers in newly produced myeloid and lymphoid cells (Bjornson, 1999, Science 283:534). Investigators at the Baylor College of Medicine have performed similar studies using satellite cells isolated from murine skeletal muscle (Jackson et al., 1999, PNAS 96:14482). When these muscle-derived cells were transplanted into lethally irradiated hosts, the investigators detected the presence of the muscle gene markers in all blood cell lineages. Together, these studies indicate that neuronal and muscle tissues contain stem cells capable of hematopoietic differentiation. This suggests that sites other than the bone marrow may provide a renewable source of hematopoietic progenitors with potential application to human disease therapy (Quesenberry et al., 1999, J. Neurotrauma 16:661: Scheffler et al., 1999, Trends Neurosci 22:348; Svendson & Smith, 1999, Trends Neurosci 22:357).
Just as neuronal and muscle cells are capable of regenerating the irradiated bone marrow, bone marrow derived cells are capable of repopulating other organ sites. When bone marrow derived hematopoietic and stromal cells are transplanted into an animal with an injured liver, they are capable of regenerating hepatic oval cells in the host animal (Petersen et al., 1999, Science 284:1168). Similarly, when labeled bone marrow stromal cells are implanted into the lateral ventricle of a neonatal mouse, they were capable of differentiating into mature astrocytes (Kopen et al., 1999, PNAS 96:10711). Indeed, when bone marrow stromal cells are transplanted intraperitoneally into mice, they are detected throughout the organs of the host animal, including the spleen, lung, bone marrow, bone, cartilage, and skin (Pereira et al., 1998, PNAS 95:1142). These studies suggest that the bone marrow stromal cell is capable of differentiating into lineages different from their original origin (Kopen et al., 1999, PNAS 96:10711).
The recent development of entire organisms from a single donor cell is consistent with this hypothesis. For example, the “Dolly” experiment showed that cells isolated from an ovine mammary gland could develop into a mature sheep. In similar murine studies, cells derived from the corpus luteum of the ovary could develop into a mature mouse. These studies suggest that stem cells with the ability to differentiate into any and all cell types continue to exist in the adult organism. Thus, “embryonic” stem cells may be retained throughout the life of an individual.
The adult bone marrow microenvironment is the potential source for these hypothetical stem cells. Cells isolated from the adult marrow are referred to by a variety of names, including stromal cells, stromal stem cells, mesenchymal stem cells (MSCs), mesenchymal fibroblasts, reticular-endothelial cells, and Westen-Bainton cells (Gimble et al., 1996, Bone 19:421). In vitro studies have determined that these cells can differentiate along multiple mesenchymal or mesodermal lineages which include, but are not limited to, adipocytes (fat cells) (Gimble et al., 1990, Eur J. Immunol 20:379), Chondrocytes (Bruder et al., 1994, J. Cell Biochem. 56:283), hematopoietic supporting cells (Pietrangeli et al., 1988, Eur. J. Immunol. 18:863), skeletal muscle myocytes (Prockop, 1998, J. Cell Biochem Suppl. 30-31:284-5), smooth muscle myocytes (Charbord et al., 2000, J. Hematother. Stem Cell Res. 9:935-43), and osteoblasts (Beresford et al., 1992, J. Cell Sci. 99:131; Dorheim et al., 1993, J. Cell Physiol. 154:317). In addition, bone marrow stromal cells display the ability to differentiate into astrocytes (Kopen et al., 1999, PNAS 96:10711) and hepatic oval cells (Petersen et al., 1999, Science 284:1168). Based on these findings, the bone marrow has been proposed as a source of stromal stem cells for regeneration of bone, cartilage, muscle, adipose tissue, liver, neuronal, and other tissues. However, extraction of bone marrow stromal cells presents a high level of risk and discomfort to the patient.
In contrast, adult human extramedullary adipose tissue-derived stromal cells represent a stromal stem cell source that can be harvested routinely with minimal risk or discomfort to the patient. Pathologic evidence suggests that adipose-derived stromal cells are capable of differentiation along multiple lineage pathways. The most common soft tissue tumors, liposarcomas, develop from adipocyte-like cells. Soft tissue tumors of mixed origin are relatively common. These tumors may include elements of adipose tissue, muscle (smooth or skeletal), cartilage, and/or bone. In patients with a rare condition known as progressive osseous heteroplasia, subcutaneous adipocytes form bone for unknown reasons.
Recent studies have demonstrated the specific ability of bone marrow-derived stromal cells to undergo neuronal differentiation in vitro (Woodbury et al., 2000, J. Neuroscience Research 61:364; Sanchez-Ramos et al., 2000, Exp. Neurology 164:247). In these investigations, treatment of bone marrow stromal cells with antioxidants, epidermal growth factor (EGF), or brain derived neurotrophic factor (BDNF) induced the cells to undergo morphologic changes consistent with neuronal differentiation, i.e., the extension of long cell processes terminating in growth cones and filopodia (Woodbury et al., 2000, J. Neuroscience Research 61:364; Sanchez-Ramos et al., 2000, Exp. Neurology 164:247). In addition, these agents induced the expression of neuronal specific protein including nestin, neuron-specific enolase (NSE), neurofilament M (NF-M), NeuN, and the nerve growth factor receptor trkA (Woodbury et al., 2000, J. Neuroscience Research 61:364; Sanchez-Ramos et al., 2000, Exp. Neurology 164:247).
Most central nervous system (CNS) injuries including stroke, trauma, hypoxia-ischemia, multiple sclerosis, seizure, infection, and poisoning, directly or indirectly involve a disruption of blood supply to the CNS. These injuries share the same common pathologic process of rapid cerebral edema leading to irreversible brain damage and eventually to brain cell death.
Stroke results in the destruction of brain tissue as a result of intracerebral hemorrhage or infarction. Stroke may be caused by reduced blood flow or ischemia that results in deficient blood supply and death of tissues in one area of the brain (infarction). The causes of ischemic stroke include blood clots that form in the blood vessels in the brain (thrombus) and blood clots or pieces of atherosclerotic plaque or other material that travel to the brain from another location (emboli). Bleeding (hemorrhage) within the brain may also cause symptoms that mimic stroke.
The CNS tissue is highly dependent on blood supply and is very vulnerable to interruption of blood supply. Even a brief interruption of the blood flow to the CNS can cause neurological deficit. The brain is believed to tolerate complete interruption of blood flow for a maximum of about 5 to 10 minutes. It has been observed that after blood flow is restored to areas of the brain that have suffered an ischemic injury, secondary hemodynamic disturbances have long lasting effects that interfere with the ability of the blood to supply oxygen to CNS tissues. Similarly, interruption of the blood flow to the spinal cord, for even short periods of time, can result in paralysis.
The majority of stroke cases are the result of ischemia (low tissue oxygen supply) due to blockages to the blood vessels serving CNS. These blockages arise from narrowing of the vessels due to build up of atherosclerotic plaques usually in combination with occlusion due to entrapment at the narrowed region of small clots of aggregated platelets (thrombus). The other cases of strokes arise from hemorrhagic (bleeding) events (e.g. intracerebral hemorrhage, subarachnoid hemorrhage) in which a blood vessel within the CNS ruptures leading to mechanical and ischemic damage.
Recognition of the “ischemic penumbra,” a region of reduced cerebral blood flow in which cell death might be prevented, has focused attention on treatments that might minimize or reverse brain damage when the treatments are administered soon after stroke onset. To date, several classes of neuroprotective compounds have been investigated for treatment of acute stroke. They have included calcium channel antagonists, N-methyl-D-aspartate (NMDA) receptor antagonists, free radical scavengers, anti-intercellular adhesion molecule 1 antibody, GM-1 ganglioside, γ-aminobutyric acid agonists, and sodium channel antagonists, among others. Results of various trials have yielded disappointing efficacy data and some evidence of safety problems, including increased mortality or psychotic effects which resulted in the early termination of the trials.
Each year about 700,000 people are afflicted by stroke in the United States. Stroke is the third highest cause of death and the leading cause of serious long term disability. The incidence of stroke increases dramatically with age with the highest risk occurring in persons at least 75 years old. Existing therapies rely on prompt treatment (i.e within minutes/hours) following a stroke but there is presently no effective or beneficial therapy that can be applied at later time points (days/weeks) after the onset of stroke. Thus there remains a large unmet need for treatments that improve neurological function after the onset of stroke.